The present invention relates generally to the modulation of the growth of collateral arteries or other arteries from preexisting arteriolar connections. In particular, the present invention provides a method for enhancing the growth of collateral arteries and/or other arteries from preexisting arteriolar connections comprising contacting tissue or cells with a monocyte chemotactic protein (MCP) or a nucleic acid molecule encoding said MCP. The present invention also relates to the use of an MCP or a nucleic acid molecule encoding said MCP for the preparation of pharmaceutical compositions for enhancing collateral growth of collateral arteries and/or other arteries from preexisting arteriolar connections. Furthermore, the present invention relates to a method for the treatment of tumors comprising contacting tissue or cells with an agent which suppresses the growth of collateral arteries and/or other arteries from preexisting arteriolar connections through the attraction of monocytes. The present invention further involves the use of an agent which suppresses the growth of collateral arteries and/or other arteries from preexisting arteriolar connections through the attraction of monocytes for the preparation of pharmaceutical compositions for the treatment of tumors.
In the treatment of subjects with arterial occlusive diseases most of the current treatment strategies aim at ameliorating their effects. The only curative approaches involve angioplasty (balloon dilatation) or bypassing surgery. The former carries a high risk of restenosis and can only be performed in certain arterial occlusive diseases, like ischemic heart disease. The latter is invasive and also restricted to certain kinds of arterial occlusive diseases. There is no established treatment for the enhancement of collateral growth.
Vascular growth in adult organisms proceeds via two distinct mechanisms, sprouting of capillaries (angiogenesis) and in situ enlargement of preexisting arteriolar connections into true collateral arteries1. Recent studies have disclosed mechanisms leading to angiogenesis with vascular endothelial growth factor (VEGF) as a major component2-6. This specific endothelial mitogen is upregulated by hypoxia and is able to promote vessel growth when infused into rabbit hindlimbs after femoral artery excision7,8. These studies however did not distinguish between capillary sprouting, a mechanism called angiogenesis, and true collateral artery growth. Whereas VEGF is only mitogenic for endothelial cells, collateral artery growth requires the proliferation of endothelial and smooth muscle cells and pronounced remodeling processes occur1,9-12. Furthermore mainly capillary sprouting is observed in ischemic territories for example in the pig heart or in rapidly growing tumors1,3,13,14. True collateral artery growth, however, is temporally and spacially dissociated from ischemia in most models studied1,15. Other or additional mechanisms as those described for angiogenesis in ischemic territories are therefore needed to explain collateral artery growth. From previous studies it is known that these collateral arteries grow from preexisting arteriolar connections1.
However, while agents such as VEGF and other growth factors are presently being employed to stimulate the development of angiogenesis after arterial occlusion, such agents are not envisaged as being capable of modulating the growth of preexisting arteriolar connections into true collateral arteries.
Thus, the technical problem of the present invention is to provide pharmaceutical compositions and methods for the modulation of the growth of collateral arteries and/or other arteries from preexisting arteriolar connections.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the invention relates to a method for enhancing the growth of collateral arteries and/or other arteries from preexisting arteriolar connections comprising contacting tissue or cells with a monocyte chemotactic protein (MCP) or a nucleic acid molecule encoding said MCP.
For the purpose of the present invention the growth of arteries from preexisting arteriolar connections is also called xe2x80x9carteriogenesisxe2x80x9d. In particular, xe2x80x9carteriogenesisxe2x80x9d is the in situ growth of arteries by proliferation of endothelial and smooth muscle cells from preexisting arteriolar connections supplying blood to ischemic tissue, tumor or sites of inflammation. These vessels largely grow outside the affected tissue but are much more important for the delivery of nutrients to the ischemic territory, the tumor or the site of inflammation than capillaries sprouting in the diseased tissue by angiogenic processes.
In the context of the present invention the term xe2x80x9cmonocyte chemotactic proteinxe2x80x9d or xe2x80x9cMCPxe2x80x9d refers to proteins and peptides which can act on monocytes and lead to augmentation of monocyte activation accumulation and migration35. Thus, according to the present invention, any MCP or other substances which are functionally equivalent to an MCP, namely which are capable of activating and attracting monocytes can be used for the purpose of the present invention. The action of the MCPs employed in the present invention may not be limited to the above-described specificity but they may also act on, for example eosinophils, lymphocyte subpopulations and/or stem cells.
In accordance with the present invention, it has been found that through the attraction of monocytes by monocyte chemotactic protein-1 (MCP-1) the growth of collateral arteries and arteriogenesis could be significantly enhanced in animals after femoral artery occlusion. Experiments performed within the scope of the present invention demonstrate that local infusion of MCP-1 increases both collateral- and peripheral conductance after femoral artery occlusion due to enhanced vessel growth by augmentation of monocyte accumulation concomitant with proliferative effects on endothelial and/or smooth muscle cells. Thus, MCPs or nucleic acid molecules encoding MCPs can be used to attract monocytes to a certain tissue or cell which in turn leads to growth of collateral arteries as well as to growth of arteries from preexisting arteriolar connections, which is needed for the cure of several occlusive diseases.
MCP-1 is a 14-kDa glycoprotein secreted by many cells, including vascular smooth muscle- and endothelial cells29-32 and induces monocyte chemotaxis at subnanomolar concentrations33. MCP-1 is a potent agonist for the xcex2 chemokine receptors CCR 2 and CCR 4 which are both mainly expressed by monocytes but also have been found to be present on basophils, T- and B-lymphocytes34. These G-protein coupled seven-transmembrane-domain receptors lead to the activation of monocytes and increased adhesiveness of integrins, a process which finally leads to monocyte arrest on endothelial cells35. The MCP-1 gene shows large interspecies homologies30 and can be induced by various cytokines (e.g. Tumor necrosis factor xcex1) and immunoglobulin G36. Recently it has been shown in vitro that gene expression and protein secretion of MCP-1 are also upregulated by shear stress and cyclic strain16-18. These mechanical forces have recently been shown to increase monocyte chemotactic protein-1 (MCP-1) secretion in cultured human endothelial cells leading to increased monocyte adhesion16-18. These findings complement the observation that monocytes adhere and migrate into the vessel wall of collateral arteries after induction of coronary artery stenosis in the dog heart at a time when the proliferation index is maximally increased19. Furthermore, monocyte accumulation is also observed in the pig microembolization model of angiogenesis20. Moreover increased levels of MCP-1 mRNA were found in ischemic tissue of microembolized porcine myocardium21 as well as in reperfused ischemic myocardium37. However, although there are several reports published that indicate that monocytes are involved in angiogenesis22-24 monocytes were not believed to play a role in the development of collateral arteries and arteriogenesis25.
The MCPs to be employed in the methods and uses of the present invention may be obtained from various sources described in the prior art; see, e.g., Proxc3x6sl69, Dahinden70, Alam71 and Oppenheim72. The potential exists, in the use of recombinant DNA technology, for the preparation of various derivatives of MCPs comprising a functional part thereof or proteins which are functionally equivalent to MCPs as described above. In this context, as used throughout this specification xe2x80x9cfunctional equivalent or xe2x80x9cfunctional partxe2x80x9d of an MCP means a protein having part or all of the primary structural conformation of an MCP possessing at least the biological property of attracting monocytes. The functional part of said protein or the functionally equivalent protein may be a derivative of an MCP by way of amino acid deletion(s), substitution(s), insertion(s), addition(s) and/or replacement(s) of the amino acid sequence, for example by means of site directed mutagenesis of the underlying DNA. Recombinant DNA technology is well known to those skilled in the art and described, for example, in Sambrook et al. (Molecular cloning; A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour N.Y. (1989)). For example, it was found that a mutation of the amino acids Leu25 and Val27 into Tyr introduces a novel monocyte chemoattractant activity into interleukin-8, which normally does not activate monocytes66.
MCPs or functional parts thereof or proteins which are functionally equivalent to MCPs, may be produced by known conventional chemical syntheses or recombinant techniques employing the amino acid and DNA sequences described in the prior art69-72, for example, MCPs may be produced by culturing a suitable cell or cell line which has been transformed with a DNA sequence encoding upon expression under the control of regulatory sequences an MCP or a functional part thereof or a protein which is functionally equivalent to MCP. Suitable techniques for the production of recombinant proteins are described in, e.g., Sambrook, supra. Methods for constructing MCPs and proteins as described above useful in the methods and uses of the present invention by chemical synthetic means are also known to those of skill in the art.
In another embodiment, the invention relates to the use of a monocyte chemotactic protein (MCP) or a nucleic acid molecule encoding said MCP for the preparation of a pharmaceutical composition for enhancing collateral growth of collateral arteries and/or other arteries from preexisting arteriolar connections.
The pharmaceutical composition comprises at least one MCP as defined above, and optionally a pharmaceutically acceptable carrier or exipient. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by conventional methods. The pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen may be determined by the attending physician considering the condition of the patient, the severity of the disease and other clinical factors. Administration of the suitable compositions may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intradermal administration.
In a preferred embodiment, said MCP used in the methods and uses of the invention is selected from the group consisting of MCP-1, MCP-2, MCP-3, MCP-4, MIP-1xcex1, RANTES, I-309 or any other CC-chemokine or classical chemoattractants like N-farnesyl peptides, C5a, leukotriene B4 or Platelet-activating factor (PAF)35,48.
In a particularly preferred embodiment, the method and uses of the invention are for the treatment of subjects suffering from occlusive disease, preferably-selected from the group consisting of coronary artery diseases, cerebral occlusive diseases, peripheral occlusive diseases, visceral occlusive diseases, renal artery disease and mesenterial arterial insufficiency.
In a further preferred embodiment, the methods and uses of the invention are for the treatment of subjects during or after exposure to an agent or radiation or surgical treatment which damage or destroy arteries.
In a preferred embodiment, the MCP used in the methods and uses of the invention is a recombinant MCP. DNA sequences encoding MCPs which can be used in the methods and uses of the invention are described in the prior art, e.g., Garcia-Zepeda34. Moreover, DNA and amino acid sequences of MCPs are available in the Gene Bank database. As described above, methods for the production of recombinant proteins are well-known to the person skilled in the art; see, e.g., Sambrook, supra.
In a further preferred embodiment, the pharmaceutical composition is designed for administration in conjugation with growth factors, preferably fibroblast growth factor or vascular endothelial growth factor (VEGF). This embodiment is particularly suited for enhancing of both sprouting of capillaries (angiogenesis) and in situ enlargement of preexisting arteriolar connections into true collateral arteries. Pharmaceutical compositions comprising, for example, an MCP such as MCP-1, and a growth factor such as VEGF may be used for the treatment of peripheral vascular diseases or coronary artery disease.
In another preferred embodiment, the method of the invention comprises
(a) obtaining cells from a subject;
(b) introducing a nucleic acid molecule encoding the MCP into said cells, thereby conferring expression and secretion of the MCP in a form suitable for the attraction of monocytes; and
(c) reintroducing the cells obtained in step (b) into the subject.
It is envisaged by the present invention that the MCPs and the nucleic acid molecules encoding the MCPs are administered either alone or in combination, and optionally together with a pharmaceutically acceptable carrier or exipient. Said nucleic acid molecules may be stably integrated into the genome of the cell or may be maintained in a form extrachromosomally. On the other hand, viral vectors may be used for transfecting certain cells or tissues, preferably cells and tissue surrounding preexisting arteriolar connections. Elements capable of targeting a nucleic acid molecule and/or protein to specific cells are described in the prior art, for example Somia, Proc. Natl. Acad. Sci., USA 92 (1995), 7570-7574. Thus, it is possible to employ the methods and uses of the invention for somatic gene therapy, which is based on introducing of functional genes into cells by ex vivo or in vivo techniques and which is one of the most important applications of gene transfer; see, e.g., Schaper73 and references cited therein.
Thus, in a preferred embodiment, the nucleic acid molecule comprised in the pharmaceutical composition for the use of the invention is designed for the expression and secretion of the MCP by cells in vivo in a form suitable for the attraction of monocytes by, for example, direct introduction of said nucleic acid molecule or introduction of a plasmid, a plasmid in liposomes, or a viral vector (e.g. adenoviral, retroviral) containing said nucleic acid molecule.
As discussed above, the growth of arteries from preexisting arteriolar connections is essential for the delivery of nutrition to tumors. Thus, if the growth of said vessels to the tumor would be suppressed suppression and/or inhibition of tumor growth is to be expected. Accordingly, the present invention also relates to a method for the treatment of tumors comprising contacting tissue or cells with an agent which suppresses the growth of collateral arteries and/or other arteries from preexisting arteriolar connections through the attraction of monocytes. Agents which suppress the growth of collateral arteries and/or other arteries from preexisting arteriolar connections may be peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, neural transmitters, peptidomimics, or PNAs (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198).
The present invention further relates to the use of an agent which suppresses the growth of collateral arteries and/or other arteries from preexisting arteriolar connections through the attraction of monocytes for the preparation of a pharmaceutical composition for the treatment of tumors.
In a preferred embodiment, the agent used in the methods and uses of the invention as described above inhibits the biological activity of a MCP and/or inhibits an intracellular signal triggered in the monocytes through the receptor for an MCP, preferably the aforementioned agent blocks and interaction of the MCP and its receptor. Various receptors of MCPs are described in the prior art, for example in Charo68 and Chemokine Receptors62. Furthermore, it has recently been shown that phosphorylation of the MCP-receptor mediates receptor desensitization and internalization and that via altering the phosphorylation sites of the receptor the chemotactic response of leukocytes to MCP-1 and related chemokines can be modulated67.
In another preferred embodiment, said receptor is selected from the group consisting of CCR1, CCR2, CCR4 and CCR5.
In a preferred embodiment, the agent which interaction of the MCP and its receptor is selected from the group consisting of
(i) an anti-MCP antibody and an anti-MCP-receptor antibody; and/or
(ii) a non-stimulatory form of an MCP protein and a soluble form of an MCP-receptor.
Anti-MCP or MCP-receptor antibodies can be prepared by well known methods using the purified MCP or its receptor or parts thereof as an antigen.
Monoclonal antibodies can be prepared, for example, by the techniques as described in Kxc3x6hler and Milstein, Nature 256 (1975), 495, and Galfrxc3xa9, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. Furthermore, antibodies or fragments thereof to the aforementioned MCPs or their receptors can be obtained by using methods which are described, e.g., in Harlow and Lane xe2x80x9cAntibodies, A Laboratory Manualxe2x80x9d, CSH Press, Cold Spring Harbour, 1988. These antibodies may be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv, or scFv fragments etc.
Non-stimulatory forms of MCPs and antagonists of MCP-receptors have been described, for example, in Gong65.
In another embodiment, the agent which suppresses the growth of collateral arteries and/or arteriogenesis is an anti-sense RNA of the MCP or of its receptor. It might be desirable to inactivate the expression of the gene encoding the MCP and/or encoding its receptor. This can be achieved by using, for example, nucleic acid molecules which represent or comprise the complementary strand of the mRNA transcript or part thereof encoding the MCP or its receptor. Such molecules may either be DNA or RNA or a hybrid thereof. Furthermore, said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotides analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. Said nucleic acid molecules may also be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell. Such nucleic acid molecules may further contain ribozyme sequences which specifically cleave the mRNA encoding the MCP or its receptor. Furthermore, oligonucleotides can be designed which are complementary to a region of the gene encoding the MCP or its receptor (triple helix; see Lee Nucl. Acids Res. 6 (1979), 3073; Cooney, Science 241 (1988), 456 and Dervan, Science 251 (1991), 1360), thereby preventing transcription and the production of the MCP or its receptor.
In a preferred embodiment, the anti-sense RNA is designed to be expressed in vascular cells or cells surrounding preexisting arteriolar connections to a tumor.
In a preferred embodiment, methods and uses of the invention are employed for the treatment of a tumor which is a vascular tumor, preferably selected from the group consisting of Colon Carcinoma, Sarcoma, Carcinoma in the breast, Carcinoma in the head/neck, Mesothelioma, Glioblastoma, Lymphoma and Meningeoma.
In a preferred embodiment, the pharmaceutical composition in the use of the invention is designed for administration by catheter intraarterial, intravenous, intraperitoneal or subcutenous routes. In the examples of the present invention the human form of the MCP-1 protein was administered locally via osmotic minipump. Positive immunohistochemical staining for BrdU infused into two animals via the same route as MCP-1 demonstrated that local delivery of substances into the collateral circulation is feasible.
The said MCP and its encoding nucleic acid molecule may be used for therapeutical purposes in various forms. Either as in the experiments described herein, locally via implanted pumps, or as arterial or venous boluses either systemically or locally via specially designed catheters or other device. They may also be injected intramuscularly or into any other tissues in which collateral artery growth needs to be promoted. Alternatively they can be bound to microcapsules or microspheres before injection.
Another approach would be to use a gene-transfer approach, either using a plasmid, or a plasmid embedded in liposomes, or viral vectors. One may either use an in vivo gene-transfer approach for which multiple devices, like double balloon or other catheters have been designed or via direct injection into the targeted tissue as described above. Alternatively it is possible to use an ex vivo approach isolating cells which are known to lodge in tissues in which vessel growth needs to be promoted or inhibited from the body which are then transfected using one of the above mentioned methods and reinjected.
The use and methods of the invention can be used for the treatment of all kinds of diseases hitherto unknown as being related to or dependent on the modulation of the growth of collateral arteries and/or other arteries from preexisting arteriolar connections. The methods and uses of the present invention may be desirably employed in humans, although animal treatment is also encompassed by the methods and uses described herein.